CXCR4 is a critical .mediator of hematopoiesis, development and organization of the immune system and as a homing receptor for stem cells. CXCR4 and its ligand, the chemokine stromal cell derived factor (SDF-1) are also implicated as key players in metastasis. CXCR4 levels on the cell surface are controlled via ubiquitin-dependent endocytosis and lysosomal degradation. I have recently determined that the deubiquitylating enzyme AMSH acts as a key effecter of CXCR4 stability and that a dominant negative form of AMSH acts to block basal turnover and stimulation-dependant degradation of CXCR4. I have raised antisera against AMSH and produced a range of mutants to determine the mechanism and cellular role of AMSH in CXCR4 trafficking and function. I propose three aims: Aim 1: Determine the role of AMSH in the stability and trafficking of CXCR4. I will utilize a set of mutants of AMSH and AMSH knock-down to determine what binding functions and regions of AMSH are required for it to act upon CXCR4 trafficking. Further studies in stable cells expressing key mutants will allow us to investigate this phenomena further and be used in aims 2 and 3. Aim 2: Determine the interactions between AMSH and adaptor/scaffold proteins that recruit AMSH to the endocytic machinery. I have determined that a specific peptide within AMSH allows it to interact with an atypical family of SH3 domains contained in the endocytic scaffold proteins Staml and Stam2 as well as adaptors of the Grb2 family. I will study these interactions biochemically and establish the role of these interactions in promoting the trafficking of CXCR4. Aim 3: Determine the effect of AMSH on CXCR4/SDF-1 mediated cell migration and chemotactic response. Using scratch-wound and transwell chemotaxis assays I will ascertain the effect of AMSH-mutant or AMSH-knock-down on functional signaling from CXCR4. Together these studies will delineate a major new modulator of CXCR4 protein levels and shed light on the endocytic trafficking of CXCR4. They will also highlight AMSH as a key control molecule in endocytic trafficking of receptors.